Inactivated bovine scours vaccines, processes and method of preventing bovine scours

ABSTRACT

Inactivated scours vaccines for immunization and protection of bovine animals from disease caused by infection with bovine rotavirus and bovine coronavirus, which comprise and effective amount of at least one inactivated viral strain are described. Polyvalent inactivated vaccines further comprising an effective amount of an antigenic component which is protective against one or more additional pathogenic organisms or viruses are also disclosed. Said vaccines are prepared from one or more strains of rota- and coronavirus,  C. perfringens  Type C bacteria and  E. coli  bacteria, and combinations thereof. Preferably, a polyvalent inactivated vaccine is provided for parenteral administration. Passive immunity is achieved in neonatal calves via immunization of pregnant cows prior to birth.

FIELD OF INVENTION

[0001] The present invention relates to the general field of immunology,and specifically to veterinary vaccines against viruses and/or bacteriathat are the causative factors in scours. More particularly, theinvention relates to the propagation of scours-causing microbialisolates and subsequent inactivation thereof, to polyvalent vaccinescontaining the inactivated scours-causing agents and to the use of suchvaccines to vaccinate bovine animals. The inactivated vaccines of thisinvention are particularly useful for vaccinating pregnant cows. Thisapplication, thus, describes neonatal scours vaccines consisting ofinactivated virus isolates and/or toxoid combinations.

BACKGROUND OF THE INVENTION

[0002] The principle of vaccination is based on two key elements ofadaptive immunity, namely specificity and memory. Memory cells allow theimmune system to mount a much stronger response on the second encounterwith antigens. This secondary response is both faster to appear and moreeffective than the primary response. The aim in vaccine development isto alter a pathogen or its toxin in such a way that they becomeinnocuous without losing antigenicity. This is possible becauseantibodies and T cells recognize particular parts of antigens, theepitopes, and not the whole organism or toxin. For example, a toxinproduced from a bacterium may be modified, e.g. by formalin treatment,so that it retains its epitopes but loses its toxicity. The resultingtoxoid is used as a vaccine. Viruses may be attenuated and/orinactivated so that they retain their antigenicity but lose theirpathogenicity.

[0003] Neonatal calf diarrhea, also known as calf scours and calfenteritis, is a serious, contagious disease caused by a variety oforganisms, including Escherichia coli, Clostridium. perfringens,rotavirus and coronavirus, often in combination and/or with otherbacteria, viruses and intestinal parasites. Although antibiotics givento scouring calves can help control bacteria, over-relying on them isineffective, as they are ineffective against viral or parasiticinfections. Moreover, antibiotics reduce the number of beneficialbacteria in the gut, and use over extended periods can lead tomicroorganisms becoming resistant to antimicrobial drugs used fortreatment, e.g. antibiotic resistant bacteria—particularly E. coli.

[0004] For many years, extensive research has been directed toward thepreparation of safe and effective veterinary scours vaccines. A numberof scours vaccines are currently marketed for use in cattle and otheranimals. These vaccines are generally classified as attenuated orinactivated, referring to the final vaccine product containing amodified live virus or a killed virus. The scours veterinary vaccinescurrently marketed include, e.g., ScourGuard™, and are known to be oflimited efficacy.

[0005] Calf scours, the leading cause of economic loss and death incalves, is usually caused by a combination of factors. Hence, a vaccineto prevent scours should include protection against the four most commoncauses of scours: coronavirus, rotavirus, E. coli and Cl. perfringensType C. Attaining high levels of antibody in the colostrum through theuse of potent vaccines has proven extremely effective in preventing calfscours. The most effective vaccination program is one in which the levelof antibodies in the dams' system peaks at or just prior to calving,providing maximum protection to the calf via the colostrum.Historically, immunization required vaccines to be given within twoweeks to a month pre-calving, and required two and sometimes threedoses. Vaccination of cows close to calving is a management problem thatcan overly stress both the cow and the fetus. The vaccines of thisinvention provide maximum protection while minimizing such stress.

[0006] Bovine coronavirus (BCV) causes both enterocolitis andrespiratory tract infections in calves and adult cattle. In someinstances, the diseases are referred to as calf diarrhea, calf scours orcalf enteritis, and winter dysentery in adult cattle. Coronaviruseffects calves as soon as their second week of life but can effect oldercalves as well. Heretofore, virus vaccines have been prepared byextensive passaging of the virulent virus. These known vaccines haveproven to be clinically ineffective against many wild-type BCVinfections. Coronavirus causes one of the most severe viral diseases ofneonatal calves and may completely destroy the villi of the intestine.Evidence also suggests that some forms of Coronavirus contribute torespiratory disease. Coronavirus causes severe disease alone, but isinfluenced by coinfections with other enteropathogens. That is,concurrent infection with E. coli and/or rotavirus often complicates thedisease process. Unlike bovine rotavirus, only one serotype of bovinecoronavirus is known.

[0007] Rotaviruses (RV) cause acute gastroenteritis, malabsorptivediarrhea, and dehydration in severe cases. The disease severity isinfluenced by coinfections with other enteropathogens. Rotavirus isrecognized as a distinct entity and is divided into six groups basedupon how the proteins on its outer surface behave antigenically. Two ofthose groups —Group A and Group B—commonly infect cattle; within thosegroups, a number of different serotypes have been identified. The mostcommon G serotypes of group A rotaviruses affecting calves are G6 andG10. G8 may also be emerging as a prevalent genotype. Three P serotypeshave been identified in calves with scours: P6(1), P7(5) and P8(11).

[0008] The two non-viral pathogens addressed by this invention includeEscherichia coli (E. coli) and Clostridium perfringens Type C (Cl.perfringens ). E. coli pathogens are commonly found in the gut and themanure of healthy cattle, resulting in most calves being exposed shortlyafter birth. These bacteria attach to the lining cells of the intestineby means of hair-like projections called pili. These attached bacteriaproduce toxins that cause the intestine to secrete large amounts offluid, which can result in scours, dehydration and death.

[0009]E. coli is divided into antigenic types based on adhesivenessfactors on the surface of the cell wall and its ability to producevarious toxins. Specific pilus formation appear to be controlled by DNAoutside the chromosome. This DNA replicates autonomously and can thus betransferred from one E. coli to the next, often resulting in increasedvirulence and mutations in the field.

[0010]Cl. perfringens Type C commonly inhabits soil as well as theintestinal tracts of healthy cattle, meaning calves are easily exposedand infected. Changes in the environment of the calf's stomach allow theType C organisms to grow, producing enterotoxins that cause severesymptoms and high death losses.

[0011] The vaccines of the present invention, accordingly, are preparedfrom virus and/or bacteria originally obtained in the field, and morespecifically, are prepared from various combinations of bovinecoronavirus, bovine rotavirus, E. coli and Cl. perfringens Type Coriginally obtained in the field. That is, they were obtained bycollecting biological samples and identifying/isolating the specificstrains.

[0012] Vaccines must contain the correct serotypes of the organismsresponsible for scours in order to prevent it. Although available scourvaccines are carefully produced to insure they contain the organismsclaimed on the label, there are differences in the number of serotypesin the products that are available. The best vaccine is one thatdemonstrates immunity in calves challenged with specific serotypes ofthe important scours-causing organisms.

[0013] Accordingly, there is a real and unsatisfied need in the art fornew treatment modalities for cattle at risk for scours-causinginfections, both in terms of a more effective and broader coveragevaccines. The novel scours vaccines disclosed herein comprise virusesthat are propagated on cell cultures and an effective vaccine isprepared by inactivating the virus after it has been grown on other cellcultures. Therefore, this invention consists of polyvalent vaccineseffective against neonatal calf scours comprising one or moreinactivated viruses, may further comprise one or more bacterial strains,and methods of preparing and administering these vaccines.

SUMMARY OF THE INVENTION

[0014] This invention relates to bovine scours vaccines, methods ofpreparing vaccines and methods of using the vaccines. These vaccines areused in healthy pregnant cattle as an aid in the prevention and controlof disease in calves caused by bovine rotavirus, bovine coronavirus,Clostridium perfringens Type C and K99 piliated Escherichia coli. Thisinvention is useful in and applicable to all conventional uses ofvaccines, in cattle and others, and derivatives thereof, but is notintended to be limited to these uses.

[0015] This invention also comprises a method of protecting newborncalves against scours by administering to pregnant cows prior to birthof said calves a vaccine prepared according to this invention.

[0016] In one embodiment of the invention, a combination bovinerotavirus and bovine coronavirus vaccine is described, said vaccineconferring heterologous coverage for multiple rotavirus and coronavirusserotypes. That is, it includes three distinct field isolates of bovinerotavirus, Group A, that encompass all of the common G and P serotypes.

[0017] In another embodiment, a combination vaccine comprising E. coliand Cl. perfringens bacterin-toxoid in combination with bovinecoronavirus and bovine rotavirus is disclosed.

[0018] Yet, another embodiment, provides vaccines comprising only bovinecoronavirus in combination with E. coli and Cl. perfringensbacterin-toxoid.

[0019] Heretofore, polyvalent scours-causing agents have not beenisolated, propagated and inactivated to produce vaccines, i.e., havingfractions comprising multiple strains of the virus and/or bacteria. Thepresent invention comprises the preparation of safe and highly effectivepolyvalent vaccines for immunization of pregnant cows in order toprovide passive immunity to neonatal calves against scours.

[0020] The vaccine of this invention is administered parenterally,preferably by intramuscular or subcutaneous injection, in one or moredoses. Preferably, a single 2.0 ml dose of vaccine containinginactivated virus-containing fluids, and may further compriseinactivated Cl. perfringens Type C fluids and inactivated E. colifluids, combined with a suitable adjuvant, and may be further combinedwith a carrier and/or stabilizer, is administered. Preferably, pregnantcows should be vaccinated one time, intramuscularly, approximately 40days or 8-10 weeks prior to calving. The virus fraction of thevaccination must be repeated in 6 weeks. With subsequent pregnancies, asingle revaccination 8-10 weeks prior to calving is required.

[0021] The coronavirus used to prepare the inactivated virus vaccines ofthis invention is the Mebus strain having ATCC Accession No. VR-874. Thebovine rotavirus used in the preparation of these vaccines includesthree distinct rotavirus strains, including Cody 81-4, G type 10B223 andB641; the former two available from and being maintained at the AnimalDisease Research and Diagnostic Laboratory, Brookings, S. Dak. by Dr.David Benfield, the remaining strain being maintained at Texas A & M byDr. Gerald Woode, Dept. of Veterinary Medicine, College Station, Tex.Four E. coli strains comprise the E. coli fraction of the presentvaccines, said E. coli strains including B41, B42, B44 and B1 17. B41was obtained from the Animal Disease Research and Diagnostic laboratory,Brookings, S. Dak. The remainder of the E. coli strains were obtainedfrom the E. coli Reference Center, Pennsylvania State University,University Park, Pa. All of the E. coli strains are maintained at the E.coli Reference Center in Pennsylvania. The Cl. perfringens Type C, is astrain identified as having genotype C, and is an alpha and beta toxinproducer, strain GL47, having ATCC accession no. ______. It was isolatedfrom a field case by Grand Laboratories, Inc. and is maintained at GrandLaboratories, Larchwood, Iowa.

[0022] To prepare the viral fractions of these inactivated vaccines, theviruses are cultivated in cell culture at from about 35° C. to about 39°C., preferably about 37° C. The virus is inactivated with aninactivating agent which does not destroy the virus particles orantigenicity according to standard methods known to the art.

[0023] To prepare the vaccine of this invention, the inactivated scourscausing agents are combined with an adjuvant., A suitable carrier and/ora stabilizer according to standard, known in the art methods may becombined therewith. Any known adjuvant that enhances the antigenicity ofthe vaccine may be used, preferably an oil based, adjuvant such asXtend® III (Grand Laboratories, Inc., Larchwood, Iowa).

[0024] It has been found that a single 2.0 ml intramuscular vaccinationwith the inactivated scours vaccine of this invention, the fractionsthereof having bovine coronavirus and bovine rotavirus titers of 10⁴ to10⁹ FAID₃₀ , Cl. perfringens antigen levels of 400 to 1000 CPU and E.coli antigen levels of 10⁴ to 10¹⁰ cfu per field dose elicitssignificant serological responses in 100% of vaccinated cattle. At 10weeks post vaccination, calves from these cattle remained protectedagainst challenge with virulent virus which effected unvaccinatedcontrols.

[0025] The inactivated scours vaccines of this invention have beenlicensed for use in cattle by the U.S. Department of Agriculture, Animaland Plant Health Inspection Services on 4 Feb. 2000 and Feb. 7, 2000 andare commercially available under the names ScourBos 4™, ScourBos6™ andScourBos9™ from Grand Laboratories, Larchwood, Iowa.

[0026] Other features and advantages of the present invention willbecome apparent from the following detailed description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

[0028] The vaccines provided herein provide ideal coverage underreal-world conditions. Requiring a limited number of doses—one per cowannually after the first year of immunization—and they providebroad-spectrum coverage against multiple serotypes of rotavirus pluscoronavirus. Three field isolates of Group A bovine rotavirus areincluded, encompassing all of the common G and P types encountered inthe United States. These vaccines show consistent characteristics andconfer consistent protection.

[0029] This invention is directed to the isolates, cell cultures,toxoids and vaccines produced therefrom, and may include variants,mutants and modifications of the same and/or similarly characterizedpathogenic agent. This invention may also relate to the use of suchisolates and vaccine combinations in producing other vaccines, e.g.,combination with other monovalent or polyvalent vaccines.

[0030] The instant invention provides vaccines having superior efficacyin relation to those currently available.

[0031] Briefly, the general production of an inactivated vaccine fromvirus begins by finding a pathogenic strain of said virus. To do so, abiological sample is obtained from sick calves and submitted to adiagnostic lab for testing, e.g., serotype determination and fieldstrain identification. The strain is then cultured and inactivated. Thepreferred inactivating agent for the viruses is β-propiolactone (BPL),as it does not alter the antigen as much as other agents, such asformalin, are prone to do. Next, a dosage is determined, i.e., 1, 2 or 5ml, and efficacy studies performed by vaccinating and subsequentlytesting efficacy by direct viral challenge and/or blood testing forseroconversion which correlates with protection.

[0032] The development of injectable bacterin-toxoid biologicals forimmunological protection is well known in the art.

[0033] Key improved characteristics by which these vaccines may include,e.g., unique and multiple serotype rotavirus isolates, unique methods ofculturing, and all vaccine fractions combinable with an oil basedadjuvant. However, one skilled in the art can readily obtain otherappropriate strains from suitable depositories, academic or commercialsources. The necessary deposits have been made under the Budapest Treatyand in accordance with MPEP §608.01(p) and 37 C.F.R. §§1.801-1.809, withthe American Type Culture Collection, 10801 University Blvd., Manassass,Va., 20110-2209 and assigned ATCC accession numbers ______. Allrestrictions on the availability to the public of the deposited materialidentified herein will be irrevocably removed upon the grant of a patenton this application, the culture(s) will be maintained for a period of30 years from the deposit date, or at least five years after the mostrecent request for a sample, whichever is longer; and the deposit willbe replaced if viable samples cannot be dispensed by the depository.

[0034] In order to prepare the vaccines of this invention, the strain isfirst cultivated and then killed, e.g., chemically inactivated, and anadjuvant added for enhancement of immunogenicity. Any of the knownadjuvants may be used, including oil based adjuvants, Freund'sincomplete adjuvant, alginate adjuvant and aluminum hydroxide adjuvant,but preferably an oil based adjuvant such as Xtend® III. The vaccinesare used in the liquid state. For optimum results, pregnant cows shouldbe immunized approximately 40 days (˜8˜10 weeks) prior to calving,followed 6 weeks later with a the appropriate viral booster, and asingle annual booster thereafter that follows the 8-10 week beforecalving guideline. When protecting calves by administering vaccine tothe pregnant cow, the present vaccine fractions should have at least arotavirus titer of 10⁷ FAID₅₀, at least a coronavirus titer of 10^(7.5)FAID₅₀, at least a Cl. perfringens antigen level of 621 CPU and at leastan E. coli antigen level of 10⁹ cfu, and preferably, a rotavirus titerof 10^(7.5) FAID₅₀, a coronavirus titer of 10⁸ FAID₅₀, a Cl. perfringensantigen level of 700 CPU and an E. coli antigen level of 10^(9.5) cfu.The vaccines should be administered parenterally, preferablyintramuscularly.

[0035] The following examples describe the preparation of theinactivated vaccines in accordance with the invention, and moreoverdescribe a manner of use thereof, but are not to be construed aslimiting the scope thereof.

[0036] Preparation of the Inactivated Vaccines

[0037] The viral agents described herein are propagated on cell linescomprising fetal bovine kidney cells. The virus may also be propagatedon cells from other tissues or cell lines of bovine or other origin.Bovine rotavirus is cultured on monolayer cell cultures that areprepared by known methods and inoculated with the viral agent.Generally, monolayer cultures are washed with a balanced salt solution(BSS) and then viral inoculum is added and allowed to be absorbed forseveral hours, e.g., 2 hours at 37° C. A maintenance medium of balancedsalt solution including antibiotic is added and the culture is incubatedat approximately 30-40° C., preferably 37° C., for approximately 1-10days. The virus is then inactivated.

[0038] An effective inactivated vaccine is prepared after inactivatingthe virus. In general, this preparation is accomplished by propagatingthe virus on, e.g., fetal bovine kidney cells, until an adequate titeris obtained. The virus is then inactivated by treating it atapproximately 20-40° C. with an inactivating agent known in the art,e.g., formalin, ethyleneimine derivatives, ultraviolet radiation orheat, and preferably β-propiolactone, for such a length of time and/orconcentration of inactivating agent as to effectively inactivate thevirus. These procedures and their details are well known in the art. Anadjuvant may be added to enhance the antigenicity. That adjuvant may beany of those known in the art, e.g., Freund's incomplete, alginate,aluminum hydroxide gel, or potassium alum, preferably an oil basedadjuvant.

[0039] Bovine coronavirus is cultured in a suspension cell culture thatis prepared by known methods and inoculated with the viral agent.

[0040] In the rota-coronavirus embodiment of the invention, a singlecoronavirus isolate is combined with three rotavirus isolates to createa polyvalent combination vaccine. The coronavirus used herein wasisolated from a calf with diarrhea. It was passaged 36 times prior tomaster seed preparation, at which time the isolate was passaged 3 timesin MDBK cells. The rotavirus strains identified as Cody 81-4 and B641were isolated from a calf with diarrhea and passaged 7 and 29 times,respectively, prior to master seed preparation, at which time the Cody81-4 isolate was passaged 1 time in MA-104 cell line and the B641isolate was passaged twice in MA-104 cells. The G type 10B223 rotavirusstrain was passaged 14 times prior to master seed preparation at whichtime the isolate was passaged 3 times in MA-104 cells.

[0041] The embodiment of the combination vaccine described hereincomprising rotavirus and coronavirus combination preferably comprisesequal parts, by volume, of each of the three strains of rotavirusisolates and equal volumes of each of the two viruses, such that theinactivated viral fluids comprise 44.4% of the total volume. In apreferred embodiment, the remainder of volume, 55.6%, is comprised of aoil based adjuvant.

[0042] More specifically, for the culture of viruses for the vaccines ofthe instant invention, cells are grown in tissue culture mediumcontaining one of the following sterile tissue culture nutrient sera andpH adjusted to 7.2 (±0.1) with 10N sodium hydroxide: Serum type % serumIron fortified calf 7.5 Fetal bovine 5.0 Bovine calf 9.0 Equine 10.0 

[0043] For virus propagation, infected cells are grown in tissue culturemedium containing 2.0% sterile tissue culture nutrient serum and trypsinsolution, the percentage of which is determined by testing the endpointdilution and the next two 2 fold dilutions for virus propagation todetermine the working dilution of the trypsin solution. For example, ifthe first dilution that shows no toxicity is 1:64, then the dilutionsfor the Virus Validation will be 1:64, 1:128 and 1:256. The use dilutionis that dilution which shows the highest geometric mean virus titer. Thespecific methodology of an optimization assay is known to the skilledartisan, i.e., within the ambit of virus research and development.Master seed and production virus is propagated in roller bottles on anapparatus rotating at 12 (±3) revolutions per hour. Seed cultures, bothfrozen and lyophilized, are stored at at least −30° C.

[0044] In use, frozen seed virus fluids of known titer are rapidlythawed to about 35-39° C., diluted with medium so as to contain therequired amount of virus, and inoculated onto the cells at the followingtarget multiplicity of infection (MOI) rates: Microorganism IsolateCulture Type MOI range (log₁₀) B. rotavirus Cody 81-4 monolayer 2.5-3.1B. rotavirus G type 10B223 monolayer 2.4-3.0 B. rotavirus B641 monolayer2.4-3.0 B. coronavirus Mebus suspension 1.7-2.3

[0045] Monolayer cultures contain 0% serum. Actual MOIs are determinedby growth studies to optimize the virus titers. Cell propagation isachieved by dispersing confluent monolayers of cells with TrypsinSolution, harvesting them in a common container and counting. Viruspropagation for rotavirus is achieved by adding virus to confluentmonolayers, allowing it to adsorb for approximately 1.5 hours andrefeeding with virus propagation medium according to known methodology.

[0046] Coronavirus propagation is affected by adding virus to suspendedcells, allowing it to adsorb for approximately 1.5 hours, adding to thevirus propagation medium and dispensing into roller bottles. Infectedcells form monolayers. All cultures are incubated at approximately35-39° C., preferably 37° C., for 1-10 days on a roller bottleapparatus.

[0047] The infected cell cultures in the roller bottles are disrupted byone freeze (≦−20° C.) thaw (35-39° C.) cycle prior to harvest. Culturesare harvested after ≧60% CPE (cytopathic effect) is observed. Actualharvest times are determined by growth studies to optimize the virustiters, but generally fall within the 1-7 day range for rotavirus and2-10 day ranges for coronavirus. Fluids to be harvested, i.e., thosecontaining the disrupted cells, are aseptically pooled in a sterilereservoir and assay samples are collected therefrom. The minimumacceptable harvest titer for rotavirus and coronavirus is 10^(4.0)FAID₅₀ per ml, preferably 10⁷ FAID₅₀ per ml and 10^(7.5) FAID₅₀,respectively.

[0048] Bovine rotavirus fluids, preferably, are inactivated withβ-propiolactone to a final concentration of 0.2% and incubated atapproximately 35-39° C., preferably 37° C., for approximately 48-54hours under constant agitation. The pH is adjusted to 7.1 (±0.2) withsterile 10N sodium hydroxide. The inactivated virus fluids are thenstored at approximately 2-70° C.

[0049] Bovine coronavirus fluids are also preferably inactivated withβ-propiolactone to a final concentration of 0.1% and incubated atapproximately 35-39° C., preferably 37° C., for approximately 22-31hours under constant agitation. The pH is adjusted to 7.1 (±0.2) withsterile 10N sodium hydroxide. The inactivated virus fluids are thenstored at approximately 2-70° C.

[0050] The vaccines of this invention may further comprise one or morepreservatives and adjuvants, e.g., preservatives may includeAmphotericin B Solution to a final concentration of 2.5 mcg/ml,penicillin/streptomycin to a final concentration of 30 units/30 mcg/mland/or thimerosal to a final concentration of 1:10,000. Completedvaccines are emulsified and comprise 55.6% adjuvant. The inactivatedfluids are combined and the pH is adjusted to 7.1 (±0.2) with sterile10N sodium hydroxide. The inactivated fluids and adjuvant are emulsifiedusing, e.g., a Ross homogenizer (Charles Ross & Son, Haupauge, N.Y.) at18 psi resulting in a flow rate of 20-22 liters per minute. Stabilizersare preferably not included in the vaccines of this invention. Sterilepreservatives are aseptically added to the viral fluids prior toemulsification and sterile antifoam is added to the aqueous pools to acompleted product concentration of 0.06%. Inactivated viral fluids maybe concentrated by ultrafiltration.

[0051] Alternative Combination Embodiments

[0052] A further aspect of this invention is the preparation and use ofcombination polyvalent vaccines comprising vaccinal amount of one ormore of the adjuvanted inactivated scours causing agents describedherein and one or more additional pathogenic entities, e.g., one or morebacteria fractions. For example, vaccines comprising vaccinal amount ofcoronavirus and/or rotavirus combined with E. coli and Cl. perfringensmay be prepared. An example of such a polyvalent bovine vaccineencompassed by this invention comprises, proportionately, approximately22.7% inactivated viral fluids, inactivated Cl. perfringens type Cfluids approximately 7.6%, approximately 14.1% inactivated E. colifluids and oil adjuvant comprising 55.6% of total vaccine volume. Moreparticularly, each 2 ml dose of prepared vaccine contains at least 10⁷FAID₅₀ of each bovine rotavirus isolate Cody 81-4, G type 10B223 andB641, 10^(7.5) FAID₅₀ of bovine coronavirus, 621 CPU of Cl. perfringensType C, and 1.6×10⁹ cfu of E. coli strains B41 and B44, and 1.0×10⁹ cfuof E. coli strains B42 and B117.

[0053] An additional embodiment of the invention is the combinationvaccine of a vaccinal amount of bovine coronavirus further comprising avaccinal amount of Cl. perfringens Type C and E. coli bacterin-toxoid,wherein the coronavirus is prepared as described supra and combined withthe Cl. perfringens Type C isolate and four E. coli isolates previouslyidentified herein. Each of the E. coli isolates was isolated from a calfwith diarrhea and a master seed established from said isolate. Theproportions of each of the E. coli isolates contained in the total E.coli fraction is, preferably, about 25%. Hence, proportionately theinactivated viral fluids comprise about 22.7%, inactivated Cl.perfringens type C fluids about 7.6%, inactivated E. coli fluids 14.1%and oil adjuvant 55.6% of total vaccine volume. More particularly, each2 ml dose of vaccine contains at least 10^(7.5) FAID₅₀ of bovinecoronavirus, 621 CPU of Cl. perfringens Type C strain GL47, and 1.6×10⁹cfu of E. coli strains B41 and B44, and 1.0×10⁹ cfu of E. coli strainsB42 and B117.

[0054] All of the embodiments of the invention have a longer duration ofprotective effects that those currently available on the market, as wellas an extremely low incidence of injection site reaction, bothintramuscular and subcutaneous. The polyvalent vaccines of thisinvention are administered parenterally, preferably by intramuscularinjection.

[0055] Use of Inactivated Vaccines

[0056] These inactivated vaccines are administered parenterally in dosesof 1 to 5 ml, preferably 2.0 ml, to pregnant cows approximately 40 daysprior to calving, to provide protection from infection with the one ormore virus and/or infection with one or more bacteria of which thevaccine is comprised.

[0057] The inactivated polyvalent vaccines were tested for effectivenessby administering the vaccine to pregnant cows approximately 40 daysprior to calving. At a later time, the calves were challenged byinoculation with virulent virus and/or bacteria. The results of thesetests are summarized in Tables 1-3.

[0058] Vaccination and Challenge

[0059] I. Bovine Coronavirus Immunogenicity

[0060] Calves born to heifers vaccinated with the inactivated polyvalentcombination vaccine of this invention comprising bovine rotavirus,bovine coronavirus, Cl. perfringens Type C-E. coli bacterin-toxoid wereprotected from a virulent challenge with bovine coronavirus. Thosecalves responded with a protective serologic responses to Cl.perfringens Type C and the K99 pilus of E. coli , as well. A single doseof vaccine is shown to protect as well as two doses.

[0061] Sixty seven Angus heifers, weighing between 500 and 800 poundswere randomly assigned into 3 test groups and pastured together prior tocalving. Three vaccines were prepared for this challenge: 1) combinationvaccine including rotavirus, coronavirus, Cl. perfringens and E. colifractions and having a coronavirus an antigenic level of 10^(7.5) FAID₅₀per 2 ml dose (“VP-760”); 2) combination vaccine including rotavirus andcoronavirus without the bacterial fractions in a 2 ml dose (“VP-761 ”);and, 3) vaccine including Cl. perfringens and E. coli without the viralfractions (1 ml dose) (“BP-635”). All animals were vaccinated with adose of one of the three vaccines, approximately 8 weeks prior tocalving. Twenty one heifers received a second viral dose approximately 6weeks thereafter.

[0062] At the time of the second vaccination, antibacterial treatmentcommenced. In particular, Aurea S700 2G was mixed in the feed at a rateto deliver 350 mg sulfamethazine and chlortetracycline per heifer perday until parturition. Individual calves received 5 ml Naxcel™ orally atbirth, followed by 5 ml Aquacillin™ intramuscularly at 1- and 2-dayspost parturition. All calves received 5 ml of Naxcel™ orally at day 17post parturition.

[0063] After calving, cow-calf pairs were separated from the heifers andhoused together until challenge. On day 3 post parturition, the cow-calfpairs were again separated and housed together for a 14-day observationperiod. The off test pairs were housed together in a separate area forat least 21 days post challenge.

[0064] Calves were challenged at 3-days post parturition with challengebovine coronavirus (UNL 4-17-92) that was obtained from and ismaintained by Dr. Gerald Duhamil at the University of Nebraska, Lincoln,Nebr. Prior to challenge, challenge doses of 2.5 ml or 5 ml virus werediluted in 5 ml Minimum Essential Medium (MEM) containing antibioticsfor total volume doses of 7.5 ml and 10.0 ml, respectively. Thechallenge dosage consisted of administration of 1 ml into each nostriland the remainder of the dosage delivered orally to the calf prior toimmediate return to the cow.

[0065] Fourteen animals were not used in the study for various reasons,none of which effected the results of the immunogenicity study. Postchallenge monitoring included clinical observation and scoring for fecalconsistency, depression and dehydration for 14-days: fecal consistencyis scored on a scale of 0-3, where 0=normal and 3=severe watery scours;dehydration is scored on a scale of 0-2, where 0=normal and 2 =sever,eyes sunken; and, depression is scored on a scale of 0-3, where 0=normaland 3=sever, unable to stand, near death.

[0066] Fecal samples were obtained from each animal daily for 14 days byinserting and withdrawing a culturette swab approximately 2 inches intothe rectum. Each swab was placed in 5.0 ml MEM with antibiotics,incubated at approximately 20-25° C. for about an hour, then centrifugedat 600×g for 20 minutes. Supernatant was poured off and frozen at −60°C. In addition, approximately 10 ml of fecal material was collected atchallenge day 0, 7 and 14, and frozen at −20° C.

[0067] Blood samples and colostrum were collected from heifers atparturition and from the cow and the calf at challenge days 0, 7 and 14and frozen at −20° C. Serum and colostrum from the heifers vaccinatedwith vaccine VP-760, and their calves, were tested for specificantitoxin titers using the procedures described in 9 CFR §113.1 1 1(c).Serum from pre-vaccination and each sampling were accordingly pooled andtested for the presence of antitoxin titers, as were the colostrumsamples. Serum were also titered for K99 antibodies using ELISAmethodology.

[0068] Serum and colostrum from the heifers vaccinated with VP-760, andtheir calves, were tested for specific antitoxin titers using theprocedures described in 9 C.F.R. §113.1 1 1 (c). A pool ofpre-vaccination serum was tested for the presence of antitoxin titers at1 International Unit (IU) for Cl. perfringens Type C. The efficacy ofthe Cl. perfringens fraction of these vaccines was demonstrated by thespecific antitoxin level passively acquired in nursing calves fromheifers vaccinated with one dose of VP-760 exceeding the requirements in9 C.F.R. §113.1 11(c) at 3 and 10 days of age. Additionally, VP-760passed the rabbit potency test.

[0069] Serum from the heifers vaccinated with VP-760 were titered forK99 antibodies using an ELISA serology test. The K99 serology assayindicated that 80 percent of the calves nursing dams with a titer of3.12 (Log Base 2) at parturition are expected to be protected, and whichdemonstrated 100% protection in calves nursing dams with a Log Base 2titer of 4 or greater at parturition. The K99 antibody titers elicitedby cattle vaccinated with VP-760 were ≧4 (Log Base 2) for 82.7% of theheifers at parturition.

[0070] The mortality rate in the control animals was 30%. None of thevaccinated animals died, regardless of which vaccination schedule theyreceived. Challenged control calves had softer than normal stools andbegan to show signs of dehydration as early as day-2 post challenge.However, there was no significant difference between the calveschallenged with 2.5 ml and 5.0 ml in the onset or overall severity atthe challenge.

[0071] Clinical signs in calves born to vaccinated heifers weresignificantly less severe (p=0.000) than those in the control calves.Control calves had clinical scores that were four times the 1 dosevaccinated animals' scores, and twice the 2 dose vaccinated animals'scores. However, the clinical scores from 1 dose vaccinated animals werenot significantly different than those from 2 dose vaccinated animals(p=0.134).

[0072] Vaccinated calves gained an average of 3.95 lbs. per day morethan challenge control calves in the first week post challenge, and 0.84lbs. per day more in the second week post challenge. Vaccinates weighedan average of 30 lbs. per calf more than controls by day 14 postchallenge. Overall, the control calves had clinical scores three-timeshigher than vaccinated calves, that data summarized in Table 1. TABLE 1AVERAGE CLINICAL SCORE CHALLENGE DOSE TOTAL OF BOTH TEST GROUP 2.5 5.0CHALLENGE GROUPS one vaccine 14.86 7.40 11.75 (n = 7) (n = 5) (n = 12)two vaccine 25.22 14.50 20.18 (n = 9) (n = 8) (n = 17) total vaccine20.69 11.77 48.23 (n = 16) (n = 13) (n = 23) Control 45.30 50.67 48.23(n = 10) (n = 13) (n = 23)

[0073] The above data establishes a minimum antigenic level of 10^(7.5)FAID₅₀ per 2 ml dose when administered subcutaneously as either a singleor two dose regiment. Moreover, the data demonstrates the lack ofinterference on the bovine coronavirus fraction by the other fractionscomprising the vaccine in toto.

[0074] All statistical analysis was performed using SYSTAT for Windows,version 5.02, clinical scores were evaluated using the Analysis ofVariance (ANOVA) test and mortality rates were evaluated by the FishersExact test.

[0075] II. Bovine Rotavirus Immunogenicity

[0076] Calves born to heifers vaccinated with the inactivated polyvalentcombination vaccines of this invention comprising bovine rotavirus,bovine coronavirus, Cl. perfringens Type C-E. coli bacterin-toxoid wereprotected from a virulent challenge with bovine rotavirus. Morespecifically, calves born to heifers vaccinated with either one or twodoses were protected from a virulent challenge with viral protein 4 (Ptypes 1 and 5) and viral protein 7 (G types 6 and 8) of Type A bovinerotavirus.

[0077] Seventy one Angus heifers, weighing between 500 and 800 pounds,were randomly assigned into 4 groups: 2 dose vaccinates, 1 dosevaccinates, controls and ScourGuard® vaccinates. The ScourGuard®vaccinates received two, 2 ml doses intramuscularly at 4 and 2 weeksprior to calving, in accordance with product label (ScourGuard 3® (K)/C,lot #149398170 manufactured by SmithKline Beecham Animal Health). Twothirds of the remainder of the cows were vaccinated with a 2 ml dose,subcutaneously, 8 weeks prior to calving with a combination vaccineincluding rotavirus, coronavirus, Cl. perfringens and E. coli fractions(“VP-916”). One third of that group, the 2 dose vaccinate group,received a second dose 6 weeks thereafter of a vaccine including onlythe rotavirus fraction (“VP-919.”). The remainder of the cows weredesignated as controls and received a single 2 ml dose of a combinationvaccine including coronavirus and bacterin fractions (“VP-917”).

[0078] Antibacterial treatment was performed as described supra.

[0079] Prior to calving all cows were pastured together. Upon calving,cow-calf pairs were separated from the herd. After challenge, the pairswere isolated to prevent “nose-to-nose” contact for approximately 12-24hours, after which the pairs were moved to test pens for the remainderof the study. Twenty-one days post challenge, the pairs were moved tooff-test pens.

[0080] Challenge material was a field strain of bovine rotavirusharvested from a 4 day old colostrum deprived Angus calf. Rotaviruspresence was detected with a Rotazyme II test kit (Abbot Laboratories).Virus isolation testing on MA104 cells was positive for rotavirus. Thetiter of the challenge material was 10^(9.22) FAID₅₀/ml ±0.64. Rotavirusonly was detected by Electron Microscopy. RNA-Page analysis identifiedthe virus as Group A rotavirus and the specific P and G types ofrotavirus were determined to be P1/P5 and G6/G8 genotypes.

[0081] After the above analysis, 5 ml of the rotavirus challengematerial was diluted in 5 ml of MEM containing antibiotics, producing a10 ml challenge dose. Calves were inoculated with the challenge dose at3-12 hours post parturition. Two 1 ml doses were delivered intranasallyand the remainder delivered orally. Subsequently, calves were monitoredand assigned daily clinical scores for fecal consistency, dehydrationand depression, as described supra. In addition, clinical scores from0-3 were assigned for respiratory signs, where 0=normal and 3=dispense,and scores for death, where 0=live and 10=dead.

[0082] Fecal samples were obtained daily for 21 days as previouslydescribed herein. Additional fecal samples were collected, as describedsupra, on challenge days 0, 7, 14 and 21. Blood samples for serum andcolostrum/milk samples were also collected, as previously described, atchallenge days 0, 7, 14 and 21. Calves weights were monitored at birthand on challenge days 0, 7, 14 and 21.

[0083] Six heifers were removed from the study prior to secondvaccination for various reasons, none of which effect the results of thestudy. Fourteen additional animals were removed from the study prior tochallenge for various reasons, none of which effected the results of thestudy. Three calves were removed from the study after challenge forvarious reasons, none of which effected the results of the study.

[0084] Challenged control calves had softer than normal stools and beganto show signs of dehydration as early as one day post challenge.Overall, the control calves had clinical scores 1.5 times higher thanthose of the calves from vaccinated heifers. Total clinical scores incalves born to vaccinated heifers were significantly lower (p=0.004)than those of the control calves. Both 2 dose and 1 dose vaccinates'scores were significantly lower than those of the control animals(p=0.006 and p=0.044, respectively). The average total clinical scoresof challenged animals are summarized below in Table 2. TABLE 2 TREATMENTAVERAGE TOTAL CLINICAL SCORE Both vaccine groups 25.79 control 39.76 2vaccines 21.60 1 vaccine 26.53 ScourGuard ® (K)/C 47.33

[0085] Fecal consistency scores of control calves were 1.5 times higherthan those of calves from vaccinated heifers. Total fecal consistencyscores from calves of vaccinated dams were significantly lower (p=0.019)than those of control calves. Two dose vaccinates scores were alsosignificantly different than the controls (p=0.01 1). However, 1 dosevaccinates' scores were not quite significant when compared to thecontrols (p=0.307).

[0086] The mortality rate in the control calves was 29%, with 60% ofthose calves being positive for rotavirus at necropsy, demonstratingclinical signs for 3-6 days prior to death and shedding virus whilealive. Thirteen percent of the one dose vaccinate calves died, while 20%of the 2 dose vaccinate calves died.

[0087] No significant weight gain difference was exhibited among thetest groups. Statistical evaluation for clinical scores, fecalconsistency scores, weight gain and mortality rates are summarized belowin Table 3. TABLE 3 All Calves Analysis Of Variance Fecal ClinicalConsistency Weight Test Group Scores Scores Day 21 Both vaccines v.controls 0.004 0.019 0.515 2 vaccines v. controls 0.006 0.011 0.082 1vaccine v. controls 0.044 0.307 0.555 ScourGuard ® (K)/C 0.839 0.3820.460

[0088] These data establish a minimum antigenic level of each strain ofrotavirus of 10⁷ FAID₅₀ per 2 ml dose.

[0089] Although the uses of the present invention have been disclosedprimarily with respect to cattle, in particular calves, this is notdeemed to limit the scope of this invention. The present invention maybe used in other fields of industry, e.g., alternate veterinaryapplications. The present invention is embodied in inactivatedpolyvalent veterinary vaccines having improved compositionalcharacteristics and improved derivative products, particularly usefulfor conferring immunity to prevent neonatal scours both in pregnant cowsand young calves. Regardless of the specific application of the instantinvention, the methodology details are calculated according to protocolswell known in the art, as well as those disclosed herein. Further, therefinement of said necessary calculations is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them without undue experimentation.

[0090] While the above description contains much specificity, thesespecificities should not be construed as limitations on the scope of theinvention, but rather exemplification of the preferred embodimentthereof. That is to say, the foregoing description of the invention isexemplary for purposes of illustration and explanation. Withoutdeparting from the spirit and scope of this invention, one skilled inthe art can make various changes and modification to the invention toadapt it to different usages and conditions. As such, these changes andmodification are properly, equitably and intended to be within the fullrange of equivalence of the following claims. Thus, the scope of theinvention should be determined by the appended claims and their legalequivalents, rather than by the examples provided herein.

We claim:
 1. An inactivated combination bovine rotavirus and coronavirusvaccine capable of inducing immunity in bovine animals without seriousside effects, the vaccine comprising a vaccinal amount of a plurality ofbovine rotavirus strains and at least one bovine coronavirus strain, andan adjuvant.
 2. The combination vaccine of claim 1 further comprising atleast one vaccinal bacteria.
 3. The combination vaccine of claim 1wherein said rotavirus strains comprise Cody 81-4, G type 10B223 andB641.
 4. The combination vaccine of claim 1 wherein said coronavirusstrain comprises the Mebus strain having ATCC accession no. VR-874. 5.The combination vaccine of claim 1 wherein said rotavirus strainscomprise Cody 81-4, G type 10B223 and B641 and the coronavirus straincomprises the Mebus strain having ATCC accession no. VR-874.
 6. Thecombination vaccine of claim 2, wherein said vaccinal bacteria comprisea vaccinal amount of a plurality of Escherichia coli bacterin strainsand at least one Clostridium perfringens Type C bacterin strain.
 7. Thecombination vaccine of claim 5 further comprising at least one vaccinalbacteria.
 8. The combination vaccine of claim 7 wherein said vaccinalbacteria comprise a vaccinal amount of a plurality of Escherichia colibacterin strains and at least one Clostridium perfringens Type Cbacterin strain.
 9. The combination vaccine of claim 6 wherein saidEscherichia coli bacterin strains comprise B41, B43, B44 and B141. 10.The combination vaccine of claim 8 wherein said Escherichia colibacterin strains comprise B41, B43, B44 and B141.
 11. The combinationvaccine of claim 6 wherein said Clostridium perfringens bacterin straincomprises GL47 having ATCC accession no _ _ _ _ _ _.
 12. The combinationvaccine of claim 8 wherein said Cl. perfringens bacterin straincomprises GL47 having ATCC accession no. _ _ _ _ _ _.
 13. Thecombination vaccine of claim 6 wherein said Escherichia coli bacterinstrains comprise B41, B43, B44 and B141 and said Clostridium perfringensbacterin strain comprises GL47 having ATCC accession no. _ _ _ _ _ _.14. The combination vaccine of claim 8 wherein said Escherichia colibacterin strains comprise B41, B43, B44 and B141 and said Clostridiumperfringens bacterin strain comprises GL47 having ATCC accession no. _ __ _ _ _.
 15. An inactivated combination vaccine capable of inducingimmunity in bovine animals without serious side effect, the vaccinecomprising a vaccinal amount of at least one bovine coronavirus strainand at least one vaccinal bacteria, said vaccinal bacteria comprising avaccinal amount of a plurality of bacterin strains, and an adjuvant. 16.The combination vaccine of claim 15 wherein said coronavirus straincomprises the Mebus strain having ATCC accession no. VR-874.
 17. Thecombination vaccine of claim 15 wherein said vaccinal bacterin comprisesa vaccinal amount of a plurality of Escherichia coli bacterin strainsand at least one Cl. perfringens Type C bacterin strain.
 18. Thecombination vaccine of claim 17 wherein said coronavirus straincomprises the Mebus strain having ATCC accession no. VR-874.
 19. Thecombination vaccine of claim 17 wherein said Escherichia coli bacterinstrains comprise B41, B43, B44 and B141.
 20. The combination vaccine ofclaim 17 wherein said Clostridium perfringens bacterin strain comprisesGL47 having ATCC accession no. ______.
 21. The combination vaccine ofclaim 17 wherein said Escherichia coli bacterin strains comprise B41B43, B44 and B141 and said Clostridium perfringens bacteria straincomprises GL47 having ATCC accession no. ______.
 22. A method ofvaccinating bovine animals comprising administering parenterally to saidanimals the combination vaccine of claims 1, 2, 5-8, 13-18 or
 21. 23.The method of claim 22 wherein the vaccine is administered byintramuscular injection.
 24. The method of claim 22 wherein the vaccineis administered by subcutaneous injection.
 25. A method of vaccinatingbovine animals comprising administering parenterally to said animals aninactivated combination bovine rotavirus and bovine coronavirus vaccinecapable of inducing immunity in bovine animals without serious sideeffect, the vaccine comprising a vaccinal amount of a plurality ofbovine rotavirus strains and at least one bovine coronavirus strain, andan adjuvant.
 26. The method of claim 25 further comprising at least onevaccinal bacteria.
 27. The method of claim 26 wherein said vaccinalbacteria comprise a vaccinal amount of a plurality of Escherichia colibacterin strains and at least one Clostridium perfringens Type Cbacterin strain.
 28. A method of vaccinating bovine animals comprisingadministering parenterally to said animals an inactivated combinationvaccine capable of inducing immunity in bovine animals without seriousside effect, the vaccine comprising a vaccinal amount of at least onebovine coronavirus strain and at least one vaccinal bacteria, saidvaccinal bacteria comprising a vaccinal amount of a plurality ofbacterin strains, and an adjuvant.
 29. The method of claim 28 whereinsaid vaccinal bacteria comprise a vaccinal amount of a plurality ofEscherichia coli bacterin strains and at least one Clostridiumperfringens Type C bacterin strain.
 30. The method of claim 25 whereinsaid rotavirus strains comprise Cody 81-4, G type 10B223 and B641. 31.The method of claim 25 wherein the coronavirus strain comprises theMebus strain having ATCC accession no. VR-874.
 32. The method of claim25 wherein the rotavirus strains comprise Cody 81-4, G type 10B223 andB641 and the coronavirus strain comprises the Mebus strain having ATCCaccession no. VR-874.
 33. The method of claim 27 wherein the Escherichiacoli bacterin strains comprise B41, B43, B44, and B141.
 34. The methodof claim 27 wherein the Clostridium perfringens bacterin straincomprises GL47 having ATCC accession no. _ _ _ _ _ _.
 35. The method ofclaim 27 wherein the Escherichia coli bacterin strains comprise B41,B43, B44, and B141 and the Clostridium perfringens bacterin straincomprises a GL47 having ATCC accession no. _ _ _ _ _ _.
 36. The methodof claim 29 wherein the coronavirus strain comprises the Mebus strainhaving ATCC accession no. VR-874.
 37. The method of claim 29 wherein theEscherichia coli bacterin strains comprise B41, B43, B44, and B141. 38.The method of claim 29 wherein the Clostridium perfringens bacterinstrain comprises GL47 having ATCC accession no. _ _ _ _ _ _.
 39. Themethod of claim 29 wherein the coronavirus strain comprises the Mebusstrain having ATCC accession no. VR-874, the Escherichia coli bacterinstrains comprise B41, B43, B44, and B141 and the Clostridium perfringensbacterin strain comprises GL47 having ATCC accession no ______.
 40. Themethod of claim 25-29, 32, 35 or 39 wherein the vaccine is administeredby intramuscular injection.
 41. The method of claim 25-29, 32, 35 or 39wherein the vaccine is administered by subcutaneous injection.
 42. Theinactivated scours vaccine of claim 1, 2, 5-8, 13-18, 21, 25-29, 32, 35or 39 wherein the virus is inactivated with an inactivating agentselected from beta-propiolactone, formalin, ethyleneimine derivatives,UV radiation and heat.
 43. The vaccine of claim 42 wherein saidinactivating agent is beta-propiolactone.
 44. The inactivated scoursvaccine of claims 1, 2, 5-8, 13-18, 21, 25-29, 32, 35 or 39 wherein theadjuvant is selected from oil based adjuvants, Freund's incomplete,alginate, aluminum hydroxide gel and potassium alum.
 45. The vaccine ofclaim 44 wherein the adjuvant is an oil based adjuvant.
 46. The vaccineof claim 42 or 44 wherein said inactivating agent comprisesβ-propiolactone and said adjuvant comprises an oil based adjuvant.
 47. Amethod of inducing scours immunity in neonatal bovine animals withoutserious side effect comprising the steps of administering the vaccine ofclaims 1, 2, 5-8, 13-18, 21, 25-29, 32, 35 or 39 to pregnant cows priorto calving.
 48. The method of claim 47 further comprising administeringa second dose of the vaccine of claims 1, 2, 5-8, 13-18, 21, 25-29, 32,35 or 39 to pregnant cows prior to calving.